Antibody-drug conjugates (ADC) are targeted anti-cancer therapeutics designed to reduce nonspecific toxicities and increase efficacy relative to conventional small molecule and antibody cancer chemotherapy. They employ the powerful targeting ability of monoclonal antibodies to specifically deliver highly potent, conjugated small molecule therapeutics to a cancer cell. To evaluate properties such as pharmacokinetics and toxicity of these antibody-drug conjugates, it is useful to be able to characterize and quantitate them from plasma, urine, and other biological samples. Additionally, the ability to quantitate the free drug (not conjugated to the antibody) in the method from the same sample and the same chromatographic injection would also be useful.
A variety of mass spectrometry techniques have been employed for identification and quantitation of small molecule therapeutics in pharmacokinetic studies, such as: electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI), fast atom bombardment (FAB), electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), and tandem mass spectrometry (MS/MS) (Yao et al (2001) Jour. of Chrom. B 752:9-16; Royer et al (1995) Rapid Comm. in Mass Spec. 9:495-502), including single ion monitoring (SIM) mode of ion selection for deconvolution (Souppart et al (2002) Jour. of Chrom. B 774:195-203; Wong et al (2001) Jour. of Chrom. 765:55-62; Yao et al (1998) Jour. of Chrom. B 718:77-85; Abdel-Hamid et al (2001) Jour. of Chrom. B 753:401-408; Marques et al (2001) Jour. of Chrom. 762:87-95). These methods and instrumentation require the separation of the various analytes from biological fluids for sufficient sensitivity. Such purification can be labor-intensive, slow, and require large volumes of sample fluids due to the low concentration of the analytes of interest in samples such as cell culture medium, human plasma, urine, and bile.
The direct combination of a separation/isolation/purification front-end step coupled with detection/characterization/quantitation by mass spectrometry is effective for metabolic studies of complex biological samples. Typically, LC/MS is used for characterization of antibodies (Martin et al (1997) Cancer Chemother. Pharmacol. 40:189-201; WO 03/046571; WO 03/046572), and ELISA is used for quantitation in biological matrices (Murray et al (2001) J. Imm. Methods 255:41-56; Kirchner et al (2004) Clin. Pharmacokinetics 43(2):83-95). ELISA assays typically are sensitive and amenable to high-throughput screens.
Recent advances in protein analysis by mass spectrometry (MS) are due to front-end gas phase ionization and introduction techniques such as electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI, US 2003/0027216) and Surface Enhanced Laser Desorption Ionization (SELDI, U.S. Pat. No. 6,020,208), as well as improvements in instrument sensitivity, resolution, mass accuracy, bioinformatics, and software data deconvolution algorithms (“Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications”, Cole, R. B., Ed. (1997) Wiley, New York; “Modern Protein Chemistry Practical Aspects”, Howard, G. C. and Brown, W. E., Eds. (2002) CRC Press, Boca Raton, Fla., p. 71-102). The primary (sequence), secondary, and tertiary structure of proteins can be probed and elucidated with MS. Electrospray ionization (ESI) provides for the atmospheric pressure ionization (API) of a liquid sample. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in the solution. An ion-sampling orifice of a mass spectrometer may be used to sample these gas phase ions for mass analysis. The response for an analyte measured by the mass spectrometer detector is dependent on the concentration of the analyte in the fluid and independent of the fluid flow rate.
Methods to detect and screening antibody-drug conjugates by Immunoaffinity membrane (IAM) capture and mass spectrometry have been disclosed (US 2005/0232929).